Antibody-drug conjugate and use thereof

Abstract

Disclosed are an anti-EGFR-cMET bispecific antibody-drug conjugate, a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof, and the use of the antibody-drug conjugate in the preparation of a drug for the treatment of EGFR and / or cMET positive diseases such as cancer.

Description

An antibody-drug conjugate and its uses Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an anti-EGFR-cMET bispecific antibody-drug conjugate, and the use of the antibody-drug conjugate in the preparation of drugs for treating diseases related to EGFR and / or cMET expression, such as cancer. Background Technology

[0002] EGFR (Epidermal Growth Factor Receptor, abbreviated as EGFR, ErbB-1, or HER1) is a member of the epidermal growth factor receptor (HER) family. It is a single-transmembrane protein composed of an extracellular domain, a transmembrane domain, and an intracellular domain. The intracellular domain contains a kinase domain and a phosphorylation site. When EGF, TGFα, etc., bind to the extracellular domain of EGFR, it causes EGFR to homodimerize or form heterodimers with other family members such as HER2 and HER3, inducing changes in the protein's three-dimensional structure and tyrosine phosphorylation. Activated EGFR can activate multiple downstream signaling pathways, such as PI3K-AKT-mTOR and Ras-Raf-MEK-ERK1 / 2, promoting cell proliferation and survival. EGFR is widely overexpressed in various epithelial-derived solid tumors, such as non-small cell lung cancer, colorectal cancer, head and neck cancer, gastric cancer, breast cancer, pancreatic cancer, renal cancer, and bladder cancer, and patients with high EGFR expression have a poor prognosis. Several EGFR-targeted therapies have been approved, such as gefitinib, erlotinib, afatinib, osimertinib, cetuximab, and amivantamab. Although they have achieved excellent clinical results, drug resistance remains a challenge. Resistance mechanisms include EGFR mutation / amplification, MET amplification, HER2 amplification, IGFR1 activation, FGFR amplification, RET / NTRK / ALK rearrangement, and EMT conversion.

[0003] cMET (Mesenchymal-Epithelial Transition Factor) is a member of the tyrosine kinase receptor family. Overexpression, amplification, and exon 14 skipping mutations lead to aberrant activation of the pathway, which is associated with tumorigenesis, progression, and drug resistance. MET overexpression occurs in various types of solid tumors, including non-small cell lung cancer, gastric cancer, colorectal cancer, breast cancer, pancreatic cancer, liver cancer, and kidney cancer. MET amplification is the most common bypass pathway in EGFR tyrosine kinase inhibitor (EGFR-TKI) resistance mechanisms. MET amplification occurs in approximately 5-30% of non-small cell lung cancer patients after first-line or later-line EGFR-TKI treatment resistance. Studies have shown that MET amplification is associated with resistance to cetuximab or panitumumab in some colorectal cancer patients. Therefore, simultaneous targeting of EGFR and MET holds promise for overcoming resistance to EGFR-targeted therapies. Antibody-drug conjugates (ADCs) have become a hot topic in anti-tumor drug development in recent years. Integrating antibodies, toxins, linkers, and conjugation technologies, they can precisely deliver cytotoxic drugs to tumor cells, thereby exerting a killing effect. ADC drugs combine the advantages of precision therapy and chemotherapy, exhibiting higher specificity and thus a higher therapeutic index compared to chemotherapy. With the development of antibody and ADC technologies, bispecific antibody ADCs have become one of the development strategies. Through the design of bispecific antibodies, it is possible to reduce on-target toxicity to normal tissues or overcome tumor heterogeneity.

[0004] Currently, several EGFR / MET bispecific antibody ADCs have entered clinical trials both domestically and internationally, but all are in early clinical stages. The product with the most progress during clinical trials is AZD9592 (tilatamig samrotecan) developed by AstraZeneca, which is in Phase 1 clinical trials. Preclinical data released by AZD9592 show that it has good anti-tumor effects in in vivo efficacy models of head and neck squamous cell carcinoma, EGFR wild-type non-small cell lung adenocarcinoma, EGFR wild-type non-small cell lung squamous cell carcinoma, and EGFR-mutant non-small cell lung cancer. Furthermore, for non-small cell lung cancer efficacy models resistant to first-line or second-line osimertinib, AZD9592 monotherapy or in combination with osimertinib can overcome osimertinib resistance.

[0005] In recent years, the field of antibody-drug conjugates (ADCs) has developed rapidly, with more than ten ADC drugs approved for marketing to treat various solid tumors or hematological malignancies. Despite achieving good efficacy, drug resistance remains a challenge. Tumor heterogeneity is one of the most pressing difficulties in cancer treatment, significantly impacting drug resistance. Currently, most ADCs are monoclonal antibody ADCs, and single-target therapy easily leads to drug resistance. EGFR overexpression is found in various solid tumors, such as lung cancer, breast cancer, colorectal cancer, head and neck cancer, gastric cancer, and pancreatic cancer, but it is also expressed in normal skin tissue, increasing the risk of target toxicity and limiting the therapeutic window. For MRG003, an EGFR monoclonal antibody ADC, the most common treatment-related adverse reactions are itching and rash. MET amplification is one of the common resistance mechanisms to EGFR inhibitors. Simultaneous targeting of EGFR and MET holds promise for overcoming EGFR inhibitor resistance. Furthermore, by modulating the affinity and valence of EGFR / MET bispecific antibody ADCs, the on-target skin toxicity of traditional EGFR monoclonal antibody ADCs can be reduced, the therapeutic window expanded, and coverage broadened to a wider range of patients. Summary of the Invention

[0006] To provide patients with more diversified treatment options, this invention has obtained a bispecific antibody-drug conjugate with higher affinity against EGFR-cMET. The antibody used in the antibody-drug conjugate of this invention is superior to Tilatamig, the antibody used in the positive control AZD9592, in terms of cell binding and endocytosis. Animal in vivo experimental data confirm that the antibody-drug conjugate obtained by this invention can more effectively inhibit the proliferation of tumor cells, and ADC-4 and ADC-5 are significantly superior to the positive control AZD9592 (ADC-6) in vivo.

[0007] In a first aspect, the present invention provides a bispecific antibody-drug conjugate against EGFR-cMET, or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, having the structure shown in formula (I):

[0008] A-(LD) d (I)

[0009] Where A is a bispecific antibody against EGFR-cMET or its antigen-binding fragment, and L is a linker part, one end of which is linked to A and the other end of which is linked to the therapeutic agent D;

[0010] d represents the molar ratio of the therapeutic agent to A, selected from an integer (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) or a decimal from 1 to 12; the therapeutic agent is a DNA topoisomerase inhibitor.

[0011] The letter d can also be understood as representing DAR, or drug-antibody conjugate ratio, which is commonly used in this field. It refers to the average number of linkers-therapeutic agents (LDs) for each A-conjugation and can be an integer or a decimal.

[0012] In some embodiments, the DNA topoisomerase inhibitor is a camptothecin analog or a DNA topoisomerase I inhibitor and its derivatives, such as DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyeclipticine, topotecan, letopotecan, belotetane, eczetane, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-pyranoxylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxy)camptothecin, etc. 2-Cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acrylamide dihydrochloride, N-[2-(dimethylamino)ethyl]-4-acrylamide or the molecule of formula D-1.

[0013] In some embodiments, the bispecific antibody against EGFR-cMET of the present invention or its antigen-binding fragment is covalently linked to a therapeutic agent via a linker.

[0014] In some implementations, L is a cuttable connector.

[0015] In some implementations, L can be cleaved under intracellular conditions or under microenvironmental conditions.

[0016] In one implementation, L is hydrolyzable at a pH less than 5.5.

[0017] In some implementations, L can be cleaved by intracellular proteases.

[0018] In some implementations, L is a cathepsin-cleavable linker.

[0019] In some implementations, L includes a dipeptide or a tetrapeptide.

[0020] In some embodiments, the dipeptide is selected from valine-citrulline (Cit) and valine-alanine (Ala), and the tetrapeptide is selected from glycine-glycine-phenylalanine (Phe)-glycine (Gly).

[0021] In some embodiments, the antibody or its antigen-binding fragment is linked to the linker via a cysteine ​​thiol group of the antibody or its antigen-binding fragment.

[0022] In one embodiment, the antibody or its antigen-binding fragment is linked to a linker via an amino group of the antibody or its antigen-binding fragment (particularly an amino group of a glutamine residue, especially a side chain amino group).

[0023] In some embodiments, L is a cleavable linker, including, but not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids, such as valine and / or citrulline, such as citrulline-valine or phenylalanine-lysine), light-labile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to avoid resistance mediated by multidrug transporters.

[0024] In some embodiments, -L- is selected from -L1-L2-L3-L4-, where L1 is a covalent linking unit covalently linked to A, L2 is an extension unit, L3 is selected from peptide residues consisting of 2-8 amino acids, and L4 is a bond or self-cleaving fragment.

[0025] In some implementations, L1 is selected from (or its open-loop form) ), The asterisk (*) indicates a connection to A.

[0026] In some implementations, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from -C 1-8 alkylene-, -C 1-8 Alkylene-C 3-8 Cycloalkylene-, -C 2-8A straight-chain or branched heteroalkylene group with 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, preferably 9-30, more preferably 9-26) carbon atoms, wherein the alkylene group, cycloalkylene group, and heteroalkylene group are each optionally independently selected from C 1-6 Alkyl groups, heteroalkyl groups with 1-6 carbon atoms (including C16 and C26) 1-6 alkoxy), hydroxy, amino, carboxyl or C 3-8 The heteroalkyl or heteroalkylene group is substituted by one or more substituents, and the heteroalkyl or heteroalkylene group contains 1-12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, preferably 1-8, more preferably 3-8) heteroatoms, each independently selected from N, O or S (preferably O).

[0027] In some implementations, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from -C 1-6 alkylene-, -C 1-3 Alkylene-C 3-6 Cycloalkyl-,-ethynyl-C 1-6 Alkylene -, -(CH2CH2O) n -、-(CH2CH2O) n C 1-3 alkylene-, -C 1-3 Alkylene (CH2CH2O) n C 1-3 alkylene-, -C 1-3 Alkylene-OC 1-3 Alkylene-, where n is an integer selected from 1 to 12 (preferably 3 to 8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0028] In some implementations, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2bEach is independently selected from -(CH2)5- and -CH2-O-CH2-.

[0029] In some implementations, L2 is selected from: -(CH2CH2O) n CH2C(O)-、-(CH2CH2O) n CH2CH2-、 The * position is connected to L1, and n is defined as above.

[0030] In some embodiments, L3 is selected from peptide residues consisting of 2-8 amino acids, each of which is independently selected from phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine. Preferably, L3 is selected from:

[0031] The * position is connected to L2.

[0032] In some implementations, L4 is selected from: bond, -*NH-CH2-, The * position is connected to L3, and R1 is selected from hydrogen and C. 1-6 alkyl.

[0033] In some implementation schemes, L is selected from:

[0034] The * position is connected to A.

[0035] In some implementation schemes, D is selected from:

[0036] Where X is selected from key, R3 and R4 are each independently selected from H, deuterium, and C. 1-3 Alkyl (e.g., methyl, ethyl, n-propyl, isopropyl), 3-6 membered cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), or R3 and R4 together with the carbon atom they are attached to form a 3-6 membered cycloalkyl (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), where n' is an integer selected from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6), and the * position is connected to L4.

[0037] In some implementations, X is selected from key,

[0038] In some implementation schemes, D is selected from:

[0039] In some embodiments, an antibody-drug conjugate having the structure shown in Formula (I) or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof is provided: Formula I has the following structure:

[0040] The definitions of R3, R4, and d are as described above.

[0041] In some embodiments, the antibody-drug conjugate of Formula I has the following structure:

[0042] , where d is defined as described above.

[0043] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention comprises an EGFR-targeting moiety and a cMET-targeting moiety. The EGFR-targeting moiety comprises a heavy chain variable region E-VH and a light chain variable region E-VL. The E-VH comprises E-HCDR1, E-HCDR2, and E-HCDR3, and the E-VL comprises E-LCDR1, E-LCDR2, and E-LCDR3. The cMET-targeting moiety comprises a heavy chain variable region M-VH and a light chain variable region M-VL. The M-VH comprises M-HCDR1, M-HCDR2, and M-HCDR3, and the M-VL comprises M-LCDR1, M-LCDR2, and M-LCDR3.

[0044] (1) The sequence of E-HCDR1 includes or is the sequence shown in SEQ ID NO.1, the sequence of E-HCDR2 includes or is the sequence shown in SEQ ID NO.2, the sequence of E-HCDR3 includes or is the sequence shown in SEQ ID NO.3, the sequence of E-LCDR1 includes or is the sequence shown in SEQ ID NO.4, the sequence of E-LCDR2 includes or is the sequence shown in SEQ ID NO.5, and the sequence of E-LCDR3 includes or is the sequence shown in SEQ ID NO.6; the sequence of M-HCDR1 includes or is the sequence shown in SEQ ID NO.11, the sequence of M-HCDR2 includes or is the sequence shown in SEQ ID NO.12, the sequence of M-HCDR3 includes or is the sequence shown in SEQ ID NO.13, the sequence of M-LCDR1 includes or is the sequence shown in SEQ ID NO.14, the sequence of M-LCDR2 includes or is the sequence shown in SEQ ID NO.15, and the sequence of M-LCDR3 includes or is the sequence shown in SEQ ID NO.16; or

[0045] (2) The sequence of E-HCDR1 includes or is the sequence shown in SEQ ID NO.21, the sequence of E-HCDR2 includes or is the sequence shown in SEQ ID NO.22, the sequence of E-HCDR3 includes or is the sequence shown in SEQ ID NO.23, the sequence of E-LCDR1 includes or is the sequence shown in SEQ ID NO.24, the sequence of E-LCDR2 includes or is the sequence shown in SEQ ID NO.25, and the sequence of E-LCDR3 includes or is the sequence shown in SEQ ID NO.26; the sequence of M-HCDR1 includes or is the sequence shown in SEQ ID NO.31, the sequence of M-HCDR2 includes or is the sequence shown in SEQ ID NO.32, the sequence of M-HCDR3 includes or is the sequence shown in SEQ ID NO.33, the sequence of M-LCDR1 includes or is the sequence shown in SEQ ID NO.34, the sequence of M-LCDR2 includes or is the sequence shown in SEQ ID NO.35, and the sequence of M-LCDR3 includes or is the sequence shown in SEQ ID NO.36. The CDR is determined according to the Kabat numbering rules.

[0046] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the EGFR targeting portion includes a heavy chain variable region E-VH and a light chain variable region E-VL, and the cMET targeting portion includes a heavy chain variable region M-VH and a light chain variable region M-VL, wherein,

[0047] (1) The sequence of E-VH includes or is the sequence shown in SEQ ID NO.7, and the sequence of E-VL includes or is the sequence shown in SEQ ID NO.8; the sequence of M-VH includes or is the sequence shown in SEQ ID NO.17, and the sequence of M-VL includes or is the sequence shown in SEQ ID NO.18; or

[0048] (2) The sequence of E-VH includes or is the sequence shown in SEQ ID NO.27, and the sequence of E-VL includes or is the sequence shown in SEQ ID NO.28; the sequence of M-VH includes or is the sequence shown in SEQ ID NO.37, and the sequence of M-VL includes or is the sequence shown in SEQ ID NO.38.

[0049] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the EGFR targeting portion includes a heavy chain variable region E-VH and a light chain variable region E-VL, and the cMET targeting portion includes a heavy chain variable region M-VH and a light chain variable region M-VL, wherein,

[0050] (1) The sequence of E-VH is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 7; the sequence of E-VL is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 8; the sequence of M-VH is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 17; the sequence of M-VL is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 18; or

[0051] (2) The sequence of E-VH is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.27, and the sequence of E-VL is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.28; the sequence of M-VH is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.37, and the sequence of M-VL is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.38.

[0052] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the anti-EGFR-cMET bispecific antibody or its antigen-binding fragment is a monoclonal antibody.

[0053] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention further includes constant region sequences for both the EGFR-targeting and cMET-targeting portions.

[0054] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the anti-EGFR-cMET bispecific antibody or its antigen-binding fragment is a full-length antibody.

[0055] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention is an isotype of IgG, IgD, IgA, IgE or IgM.

[0056] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention is an IgG1, IgG2, IgG3 or IgG4 subtype.

[0057] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention is an IgG1 subtype.

[0058] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the anti-EGFR-cMET bispecific antibody or its antigen-binding fragment is an antigen-binding fragment selected from Fv, scFv, Fab, Fab' or (Fab')2.

[0059] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the EGFR-targeting portion and the cMET-targeting portion are chimeric, humanized, or fully human antibodies or their antigen-binding fragments, preferably fully human antibodies or their antigen-binding fragments.

[0060] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention is an IgG-like antibody, the structure of which is shown in Figure 8.

[0061] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the EGFR targeting portion includes a first heavy chain and a first light chain, and the cMET targeting portion includes a second heavy chain and a second light chain. The first heavy chain includes a heavy chain variable region E-VH, and the first light chain includes a light chain variable region E-VL. The E-VH includes E-HCDR1, E-HCDR2, and E-HCDR3, and the E-VL includes E-LCDR1, E-LCDR2, and E-LCDR3. The second heavy chain includes a heavy chain variable region M-VH, and the second light chain includes a light chain variable region M-VL. The M-VH includes M-HCDR1, M-HCDR2, and M-HCDR3, and the M-VL includes M-LCDR1, M-LCDR2, and M-LCDR3.

[0062] (1) The sequence of E-HCDR1 includes or is the sequence shown in SEQ ID NO.1, the sequence of E-HCDR2 includes or is the sequence shown in SEQ ID NO.2, the sequence of E-HCDR3 includes or is the sequence shown in SEQ ID NO.3, the sequence of E-LCDR1 includes or is the sequence shown in SEQ ID NO.4, the sequence of E-LCDR2 includes or is the sequence shown in SEQ ID NO.5, and the sequence of E-LCDR3 includes or is the sequence shown in SEQ ID NO.6; the sequence of M-HCDR1 includes or is the sequence shown in SEQ ID NO.11, the sequence of M-HCDR2 includes or is the sequence shown in SEQ ID NO.12, the sequence of M-HCDR3 includes or is the sequence shown in SEQ ID NO.13, the sequence of M-LCDR1 includes or is the sequence shown in SEQ ID NO.14, the sequence of M-LCDR2 includes or is the sequence shown in SEQ ID NO.15, and the sequence of M-LCDR3 includes or is the sequence shown in SEQ ID NO.16; or

[0063] (2) The sequence of E-HCDR1 includes or is the sequence shown in SEQ ID NO.21, the sequence of E-HCDR2 includes or is the sequence shown in SEQ ID NO.22, the sequence of E-HCDR3 includes or is the sequence shown in SEQ ID NO.23, the sequence of E-LCDR1 includes or is the sequence shown in SEQ ID NO.24, the sequence of E-LCDR2 includes or is the sequence shown in SEQ ID NO.25, and the sequence of E-LCDR3 includes or is the sequence shown in SEQ ID NO.26; the sequence of M-HCDR1 includes or is the sequence shown in SEQ ID NO.31, the sequence of M-HCDR2 includes or is the sequence shown in SEQ ID NO.32, the sequence of M-HCDR3 includes or is the sequence shown in SEQ ID NO.33, the sequence of M-LCDR1 includes or is the sequence shown in SEQ ID NO.34, the sequence of M-LCDR2 includes or is the sequence shown in SEQ ID NO.35, and the sequence of M-LCDR3 includes or is the sequence shown in SEQ ID NO.36. The CDR is determined according to the Kabat numbering rules.

[0064] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the first heavy chain includes a heavy chain variable region E-VH, the first light chain includes a light chain variable region E-VL, the second heavy chain includes a heavy chain variable region M-VH, and the first light chain includes a light chain variable region M-VL, wherein...

[0065] (1) The sequence of E-VH includes or is the sequence shown in SEQ ID NO.7, and the sequence of E-VL includes or is the sequence shown in SEQ ID NO.8; the sequence of M-VH includes or is the sequence shown in SEQ ID NO.17, and the sequence of M-VL includes or is the sequence shown in SEQ ID NO.18; or

[0066] (2) The sequence of E-VH includes or is the sequence shown in SEQ ID NO.27, and the sequence of E-VL includes or is the sequence shown in SEQ ID NO.28; the sequence of M-VH includes or is the sequence shown in SEQ ID NO.37, and the sequence of M-VL includes or is the sequence shown in SEQ ID NO.38.

[0067] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the first heavy chain includes a heavy chain variable region E-VH, the first light chain includes a light chain variable region E-VL, the second heavy chain includes a heavy chain variable region M-VH, and the second light chain includes a light chain variable region M-VL, wherein...

[0068] (1) The sequence of E-VH is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 7; the sequence of E-VL is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 8; the sequence of M-VH is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 17; the sequence of M-VL is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 18; or

[0069] (2) The sequence of E-VH is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.27; the sequence of E-VL is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.28; the sequence of M-VH is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.37; and the sequence of M-VL is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.38.

[0070] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the anti-EGFR-cMET bispecific antibody or its antigen-binding fragment is a monoclonal antibody.

[0071] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention further includes constant region sequences for both the EGFR-targeting and cMET-targeting portions.

[0072] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the anti-EGFR-cMET bispecific antibody or its antigen-binding fragment is a full-length antibody.

[0073] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention is an isotype of IgG, IgD, IgA, IgE or IgM.

[0074] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention is an IgG1, IgG2, IgG3 or IgG4 subtype.

[0075] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention is an IgG1 subtype.

[0076] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention includes or has ADCC, ADCP or CDC effects.

[0077] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention includes or has a weakened or eliminated ADCC, ADCP or CDC effect.

[0078] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention comprises or has one or more mutations selected from IgG1 L234F, L235E, or P331S. Unless otherwise specified, the mutated IgG1 L234F, L235E, or P331S targets a sequence in the constant region of the human IgG1 heavy chain.

[0079] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention includes or has an IgG1 L234F-L235E-P331S three-point mutation.

[0080] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention comprises or has an IgG1 L234F-L235E-P331S three-point mutation in the first and second heavy chains of the anti-EGFR-cMET bispecific antibody.

[0081] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention includes or has a mutation that prolongs the half-life of the anti-EGFR-cMET bispecific antibody.

[0082] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention includes or has a Knob-Hole mutation.

[0083] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the first heavy chain of the anti-EGFR-cMET bispecific antibody includes or has a Hole mutation, and the second heavy chain includes or has a Knob mutation.

[0084] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the first heavy chain of the anti-EGFR-cMET bispecific antibody includes or has a Knob mutation, and the second heavy chain includes or has a Hole mutation.

[0085] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the first heavy chain of the anti-EGFR-cMET bispecific antibody includes or has an IgG1 T366S-L368A-Y407V-Y349C mutation, and the second heavy chain includes or has an IgG1 T366W-S354C mutation.

[0086] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the first heavy chain of the anti-EGFR-cMET bispecific antibody includes or has an IgG1 T366W-S354C mutation, and the second heavy chain includes or has an IgG1 T366S-L368A-Y407V-Y349C mutation.

[0087] In this invention, the heavy chain constant region numbering is determined using Kabat's EU index.

[0088] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention comprises a κ-type or λ-type light chain constant region in the first and second light chains of the anti-EGFR-cMET bispecific antibody.

[0089] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the first light chain of the anti-EGFR-cMET bispecific antibody includes a λ-type light chain constant region, and the second light chain includes a κ-type light chain constant region.

[0090] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the second heavy chain and the second light chain of the anti-EGFR-cMET bispecific antibody exhibit CH1-CL exchange.

[0091] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the first heavy chain and the first light chain of the anti-EGFR-cMET bispecific antibody exhibit CH1-CL exchange.

[0092] In some embodiments, in the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention, the first heavy chain, the first light chain, the second heavy chain, and the second light chain are chimeric, humanized, or fully human antibodies or their antigen-binding fragments, preferably fully human antibodies or their antigen-binding fragments.

[0093] In some embodiments, the anti-EGFR-cMET bispecific antibody-drug conjugate of the present invention,

[0094] (1) The first heavy chain includes or has the sequence shown in SEQ ID NO. 9, the first light chain includes or has the sequence shown in SEQ ID NO. 10, the second heavy chain includes or has the sequence shown in SEQ ID NO. 19, and the second light chain includes or has the sequence shown in SEQ ID NO. 20; or

[0095] (2) The first heavy chain includes or has the sequence shown in SEQ ID NO.29, the first light chain includes or has the sequence shown in SEQ ID NO.30, the second heavy chain includes or has the sequence shown in SEQ ID NO.39, and the second light chain includes or has the sequence shown in SEQ ID NO.40.

[0096] In some implementations, the anti-EGFR-cMET bispecific antibody-drug conjugate is ADC-1, ADC-2, ADC-3, ADC-4, or ADC-5.

[0097] Secondly, the present invention provides an isolated nucleic acid molecule encoding an anti-EGFR-cMET bispecific antibody comprising a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein...

[0098] (1) The first heavy chain includes or has the sequence shown in SEQ ID NO. 9, the first light chain includes or has the sequence shown in SEQ ID NO. 10, the second heavy chain includes or has the sequence shown in SEQ ID NO. 19, and the second light chain includes or has the sequence shown in SEQ ID NO. 20; or

[0099] (2) The first heavy chain includes or has the sequence shown in SEQ ID NO.29, the first light chain includes or has the sequence shown in SEQ ID NO.30, the second heavy chain includes or has the sequence shown in SEQ ID NO.39, and the second light chain includes or has the sequence shown in SEQ ID NO.40.

[0100] In some embodiments, the present invention provides a combination of isolated polynucleotides comprising a light chain polynucleotide encoding a light chain of the second aspect of the anti-EGFR-cMET bispecific antibody or an antigen-binding fragment thereof, and a heavy chain polynucleotide encoding the second aspect of the anti-EGFR-cMET bispecific antibody or an antigen-binding fragment thereof. In some embodiments, the polynucleotide is operatively linked to a regulatory sequence that can be recognized by host cells transformed with the vector.

[0101] Thirdly, the present invention provides a carrier containing the nucleic acid molecules described in the second aspect.

[0102] In some embodiments, the expression vector of the present invention comprises a nucleic acid molecule or a combination of nucleic acid molecules as described in the second aspect of the present invention, said nucleic acid molecule being efficiently linked to a regulatory sequence that allows the polypeptide it encodes to be expressed in a host cell or cell-free expression system. The choice of expression vector depends on the choice of host cell and can be selected to achieve the desired expression and regulatory characteristics in the selected host cell.

[0103] The nucleic acid in the vector can be operatively linked to one or more expression control sequences. As used herein, "operatively linked" means incorporated into the genetic construct such that the expression control sequence effectively controls the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence consisting of a region of the DNA molecule typically located within 100 nucleotides upstream of the transcription start site (usually near the start site of RNA polymerase II). For the coding sequence to be under the control of the promoter, the translation start site of the polypeptide translation reading frame must be located between 1 and 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function at different distances from the transcription start site. Enhancers can also be located downstream of the transcription start site. When RNA polymerase is able to transcribe the coding sequence into mRNA, and then the mRNA can be translated into the protein encoded by the coding sequence, the coding sequence is "operatively linked" to and "under the control" of the expression control sequence in the cell.

[0104] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophages, baculoviruses, tobacco mosaic virus, herpesviruses, cytomegaloviruses, retroviruses, vaccinia virus, adenoviruses, and adeno-associated viruses. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0105] Expression vectors may include tag sequences. Tag sequences are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted at any position within the polypeptide, including the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, Fc fragments, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, Flag™ tags (Kodak, New Haven, CT), maltose E-binding protein, and protein A. In some embodiments, the nucleic acid molecule encoding the EGFR-cMET fusion polypeptide is contained in a vector of nucleic acid containing one or more domains encoding the constant region of the Ig heavy chain, such as amino acid sequences corresponding to the hinge region, CH2 region, and CH3 region of the human immunoglobulin Cγ1 chain (Fc fragment).

[0106] Fourthly, the present invention provides a host cell containing the nucleic acid molecule described in the second aspect or the vector described in the third aspect.

[0107] In some embodiments, the host cell can be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. Prokaryotic host cells can be *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*, etc. Eukaryotic host cells can be fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*, insect cells such as *Ardisia crenata*, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the host cell is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell, or COS cell.

[0108] Fifthly, the present invention provides a pharmaceutical composition comprising the aforementioned anti-EGFR-cMET bispecific antibody-drug conjugate or its tautomer, stereoisomer or pharmaceutically acceptable salt thereof.

[0109] In this invention, the pharmaceutical composition according to the fifth aspect further includes a pharmaceutically acceptable carrier.

[0110] In a sixth aspect, the present invention provides the use of the aforementioned bispecific antibody-drug conjugate against EGFR-cMET or its tautomers, stereoisomers or pharmaceutically acceptable salts or the aforementioned pharmaceutical compositions in the preparation of a medicament for treating tumors.

[0111] In some implementations, the tumor is a tumor whose tumor cells express EGFR and / or cMET on their surface.

[0112] In some implementations, the tumor is an EGFR and / or cMET positive tumor.

[0113] In some implementations, the tumor is a solid tumor.

[0114] In some implementations, the tumor is a hematologic malignancy.

[0115] In some implementations, the tumor is a malignant tumor.

[0116] In some implementations, the tumor is cancer.

[0117] In some implementations, the tumor is lung cancer, stomach cancer, colorectal cancer, or skin cancer.

[0118] In some implementations, the tumor is lung adenocarcinoma, gastric cancer, colorectal cancer, or skin cancer.

[0119] In a seventh aspect, the present invention provides a method for treating tumors in an individual, particularly tumors on which tumor cells express EGFR and / or cMET, the method comprising administering to the individual a therapeutically effective amount of the aforementioned anti-EGFR-cMET bispecific antibody-drug conjugate or its tautomer, stereoisomer, or pharmaceutically acceptable salt or the aforementioned pharmaceutical composition.

[0120] In some implementations, the tumor is an EGFR and / or cMET positive tumor.

[0121] In some implementations, the tumor is a solid tumor.

[0122] In some implementations, the tumor is a hematologic malignancy.

[0123] In some implementations, the tumor is a malignant tumor.

[0124] In some implementations, the tumor is cancer.

[0125] In some implementations, the tumor is lung cancer, stomach cancer, colorectal cancer, or skin cancer.

[0126] In some implementations, the tumor is lung adenocarcinoma, gastric cancer, colorectal cancer, or skin cancer.

[0127] Eighthly, the present invention provides the use of a therapeutically effective amount of the aforementioned anti-EGFR-cMET bispecific antibody-drug conjugate or its tautomer, stereoisomer or pharmaceutically acceptable salt or the aforementioned pharmaceutical composition for treating tumors in an individual, particularly tumors on which EGFR and / or cMET are expressed on the surface of tumor cells.

[0128] In some implementations, the tumor is an EGFR and / or cMET positive tumor.

[0129] In some implementations, the tumor is a solid tumor.

[0130] In some implementations, the tumor is a hematologic malignancy.

[0131] In some implementations, the tumor is a malignant tumor.

[0132] In some implementations, the tumor is cancer.

[0133] In some implementations, the tumor is lung cancer, stomach cancer, colorectal cancer, or skin cancer.

[0134] In some implementations, the tumor is lung adenocarcinoma, gastric cancer, colorectal cancer, or skin cancer.

[0135] definition

[0136] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0137] Although the numerical ranges and parameter approximations shown in the broad scope of this invention are intended to be approximated, the values ​​described in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the stated range “1 to 10” should be considered to include any and all subranges between the minimum value 1 and the maximum value 10 (inclusive); that is, all subranges beginning with a minimum value of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum value of 10 or less, such as 5.5 to 10. Additionally, any references marked “incorporated herein” should be understood to be incorporated herein in their entirety.

[0138] The term “individual” as used in this article refers to mammals, such as humans, but can also refer to other animals, such as wild animals, livestock, or laboratory animals (e.g., chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0139] As used herein, the term "antigen" refers to a predetermined target to which an antibody can selectively bind. Examples of antigens include, but are not limited to, peptides, sugars, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds.

[0140] In a broad sense, an "antibody" can refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Therefore, it encompasses complete antibodies / full-length antibodies, single-chain antibodies, or any antigen-binding fragment of an antibody (also known as an "antigen-binding moiety"). When "antibody" and "antigen-binding fragment / antigen-binding moiety" appear in the same context, "antibody" can be understood as the complete entity relative to the "antigen-binding fragment / antigen-binding moiety," and both correspond to the broad concept of antibody.

[0141] The term "anti-EGFR-cMET bispecific antibody" includes antibodies that can bind to EGFR and cMET with sufficient affinity, making the antibody usable as a diagnostic and / or therapeutic agent when targeting EGFR and cMET.

[0142] A "full-length antibody" refers to a protein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into multiple highly variable regions called complementarity-determining regions (CDRs), interspersed with multiple more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but the antibody does not need to belong to any specific class. Immunoglobulins can be designated into different classes based on the antibody amino acid sequence of the constant region of the heavy chain. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant regions of the heavy chain corresponding to different immunoglobulin classes are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known. Chimeric or humanized antibodies are also included in the antibodies according to the present invention. It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. There are several common ways to define the CDR amino acid sequence of VH or VL, such as the Kabat definition, the IMGT definition, and the Chothia definition. For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the VH and VL amino acid sequences can usually be determined according to different definitions. In the embodiments of this invention, the Kabat definition of the CDR amino acid sequence is used. For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the variable region amino acid sequence can be analyzed in various ways.

[0143] The term "humanized antibody" refers to an antibody obtained by grafting a CDR sequence derived from another mammalian species, such as a mouse line, onto a human framework sequence. To preserve binding affinity, some residues in the backbone (called FR) segment can be modified. Humanized antibodies or fragments thereof according to the present invention can be prepared using techniques known to those skilled in the art.

[0144] The term "chimeric antibody" refers to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species, for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody. Chimeric antibodies or fragments thereof according to the invention can be prepared using gene recombination technology. For example, the chimeric antibody can be produced by cloning recombinant DNA containing a promoter and a sequence encoding the variable region of a non-human, particularly mouse, monoclonal antibody according to the invention, and a sequence encoding the constant region of a human antibody. The chimeric antibody of the invention encoded by such a recombinant gene will be, for example, a mouse-human chimera, whose specificity is determined by the variable region derived from mouse DNA and whose isotype is determined by the constant region derived from human DNA.

[0145] The term "monoclonal antibody" refers to an antibody obtained from a population of essentially homogeneous antibodies, meaning that the individual antibodies that make up the population are identical, except that naturally occurring mutations may exist in a small number of individuals.

[0146] As used herein, the terms “antigen-binding fragment” or “antigen-binding portion” or “antigen-binding region” are used interchangeably to refer to a portion of an antibody containing amino acid residues that interact with the antigen and confer specificity and affinity to the antigen, particularly antibody fragments such as Fv, Fab, F(ab')2, or Fab', optionally containing chemically modified fragments that add half-life, such as the addition of poly(alkylene) glycols like polyethylene glycol (“PEGylated”) (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) (“PEG” stands for polyethylene glycol), which has EGFR-cMET binding activity. Preferably, the antigen-binding fragment will consist of or contain a portion of the heavy or light chain variable chain of its source antibody, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, and such antigen-binding fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids of its source antibody sequence. Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab')2 fragments, which may be divalent fragments having two Fab' fragments connected by disulfide bridges (i.e., Fab' dimers) in the hinge region; (3) Fv fragments having a single arm of antibody with VL and VH domains; and (4) VHH fragments consisting of VH domains.

[0147] The term "single-chain antibody (scFv)" refers to a single polypeptide chain consisting of VH and VL domains linked by peptide linkers. (scFv)2 contains two VH domains and two VL domains linked by peptide linkers, with the two VL domains combined with the two VH domains via disulfide bridges.

[0148] The terms “Fc fragment,” “Fc region,” “Fc domain,” “Fc part,” or similar terms refer to a portion of the constant region of the antibody heavy chain, including the hinge region, the CH2 segment, and the CH3 segment of the constant region. The Fc region of an anti-EGFR-cMET bispecific antibody can be engineered or modified, including effector-related modifications, to reduce or eliminate antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), for example, by introducing one or more amino acid substitutions / mutations into the Fc region of the antibody.

[0149] As used in this article, "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

[0150] The term "bispecific antibody" refers to an antibody that simultaneously binds to two antigenic epitopes. These two epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can consist of two Fc fragments and two antigen-binding fragments fused to them, with a structure similar to natural antibodies, the difference being that the two arms bind different antigenic targets or epitopes; this is also called an IgG-like bispecific antibody. Non-IgG-like bispecific antibodies generally use scFv fragments or Fab fragments as basic modules. By utilizing appropriately modified peptide linkers, scFv fragments or Fab fragments can form dimers, trimers, tetramers, pentamers, or even higher-order oligomers.

[0151] Generally, to prepare monoclonal antibodies or their functional fragments, especially murine monoclonal antibodies or their functional fragments, one can refer to the techniques described in particular in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or refer to the techniques described by Kohler and Milstein for preparation from hybridoma cells (Nature, 256: 495-497, 1975).

[0152] In describing some embodiments involving amino acid or nucleic acid sequence mutations, the present invention uses the designation "XaaaY" (e.g., L234A), where "aaa" represents the sequence position of the amino acid or base (when a specific reference sequence exists, "aaa" represents the residue order position in that reference sequence; or it can be numbered according to the position numbering system commonly used in the art, such as the EU numbering system), "X" represents the original amino acid or base at "aaa", and "Y" represents the changed amino acid or base at "aaa". As an example, when describing the L234A mutation in the human heavy chain constant region, it means that, according to the EU numbering system for the human heavy chain constant region, leucine (L) at position 234 is mutated to alanine (A).

[0153] The term "isolated" refers to biological components (e.g., nucleic acids, proteins (including antibodies), or organelles) that have been substantially separated from or purified from other biological components (i.e., other and extra chromosomal DNA and RNA, proteins, and organelles) in their natural environment (e.g., cells). "Isolated" nucleic acids and proteins include those purified using standard purification methods. The term also includes nucleic acids and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0154] An "expression vector" is a vector that includes one or more expression control sequences. An "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0155] The term "antibody-drug conjugate" or "antibody-conjugated drug" (ADC) refers to the linking of a target ligand, such as an antibody (e.g., a monoclonal antibody), or an antibody fragment to a biologically active molecule via a linker or adapter.

[0156] Unless otherwise specified, the terms "pharmaceutically acceptable salt" or "medicinal salt" refer to a salt suitable for contact with mammalian, particularly human, tissues without excessive toxicity, irritation, allergic reactions, etc., within the limits of reasonable medical judgment, and in accordance with a reasonable benefit / risk ratio. Medically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salts can be prepared in situ during the final isolation and purification of the compounds of this invention, or solely by reacting a free base or free acid with a suitable reagent. Exemplary salts include, but are not limited to: sulfates, trifluoroacetates, citrates, acetates, oxalates, hydrochlorides, hydrobromides, hydroiodates, nitrates, hydrogen sulfates, phosphates, acidic phosphates (-H₂PO₄) salts, phosphites, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, hydrogen tartrates, ascorbic acid salts, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, potassium salts, sodium salts, ammonium salts, calcium salts, etc. Additionally, pharmaceutically acceptable salts have more than one charged atom in their structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counter atoms. For example, a pharmaceutically acceptable salt has one or more charged atoms and / or one or more counter atoms.

[0157] Unless otherwise specified, the compounds described in this invention (including antibody-drug conjugates) also contain "isotope labels," which refer to compounds of this invention that can exist in an isotopically traced or enriched form, containing one or more atoms whose atomic weight or mass number differs from the atomic weight or mass number of the most abundant atoms found in nature. The isotope can be radioactive or non-radioactive. Commonly used isotopes for isotope labeling include hydrogen isotopes. 2 H and 3 H; Carbon isotopes: 13 C and 14 C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 123 I and 125 I; Nitrogen isotopes: 13 N and15 N; oxygen isotopes: 15 O, 17 O and 18 O and sulfur isotopes 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Especially 2 H and 13 C, because they are easy to label and convenient to detect, are more widely used. Some heavy isotopes, such as deuterium (… 2 Substitution with H can enhance metabolic stability and prolong the half-life, thereby achieving the goal of reducing dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized by starting with a labeled starting material, using known synthetic techniques similar to those used to synthesize non-isotope-labeled compounds.

[0158] Unless otherwise specified, the compounds described in this invention (including antibody-drug conjugates) also comprise "solvates," where "solvate" refers to the physical association of the compound of this invention with one or more solvent molecules (whether organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. Solvent molecules in a solvate may be present in a regular and / or disordered arrangement. Solvates may contain stoichiometric or non-stoichiometric solvent molecules. "Solvate" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0159] Unless otherwise specified, the term "stereoisomer" refers to compounds having the same chemical structure but with different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and inhibited isomers. Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0160] The term "linker-drug" refers to the partial structure of an "antibody-drug conjugate" consisting of a linker and a bioactive compound.

[0161] The term "linker" refers to a chemical structural fragment, denoted by L, that is linked at one end to an antibody and at the other end to a cytotoxic drug. In some embodiments of the invention, the "linker" is formed by linking an antibody to a "linker-drug compound." In other embodiments, a portion of the "linker" is first linked to an antibody, and another portion is linked to a bioactive compound, and then the linker is formed by covalently linking the different portions of the linker. The linker of the present invention comprises a covalently linked unit linked to an antibody, an extension unit, peptide residues, and optionally a self-cleaving fragment.

[0162] In this invention, the linker-drug compound and the antibody are linked via conventional coupling methods in the art, including lysine coupling, inter-light and heavy chain reducing disulfide bond coupling, and directional coupling. Preferably, this invention uses inter-light and heavy chain reducing disulfide bond coupling, i.e., the coupling is achieved by reacting one or more of the disulfide bond sites between the light and heavy chains (two sites between the heavy chain and two sites between the heavy and light chains) with a thiol group (sulfur atom of a cysteine ​​residue) formed after reduction. In this invention, the linker L and the antibody are denoted as L1, which is formed by the reaction of the L1' group in the linker compound with the antibody, for example... or The asterisk (*) indicates a link to the thiol group of the antibody, and the doublet (**) indicates a link to the spacer.

[0163] The term "extension unit" refers to the linking group between the covalent linker unit in linker L and the polypeptide sequence. This linker can be any divalent organic group, such as a chemical bond, C... 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-10 cycloalkyl, C 6-12 aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic, or a combination of two or more of these groups; the C 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-10 cycloalkyl, C 6-12 Aryl, 5-12 membered heteroaryl, 5-12 membered heterocyclic, or a combination of two or more of these groups, optionally separated by carbonyl, O, S, or N atoms; the C 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-10 cycloalkyl, C 6-12 Aryl, 5-12-membered heteroaryl, and 5-12-membered heterocyclic groups can be optionally C-shaped. 1-6 Alkyl, C 3-6 Cycloalkyl, halogen atom, halogenated C 1-6Alkyl substitution; preferably, the extension unit comprises hydrophilic segment substitution or insertion; preferably, the extension unit is as defined in L2.

[0164] The term "peptide residue" is well known in the art and is selected from a divalent peptide group comprising 2 to 8 optionally substituted protein or non-protein amino acids, L-type or D-type amino acid residues, each of which may be the same or different and is independently selected from residues of the following amino acids: alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), pentaneous (Nva), or leucine (Nle), or analogues of the above amino acids. The peptide residues in this invention may contain substituted or derived amino acid residues, such as amino acid residues with side-chain amino groups specifically substituted as defined in CN115279417A, for example:

[0165] The term "self-cleaving fragment" is well known in the art, such as the p-aminobenzyl carbonate fragment or -NH-CH2- fragment commonly used in the art. In specific embodiments of the present invention, the "self-cleaving fragment" includes the modification of "hydrophilic fragment".

[0166] The term "hydrophilic fragment" is well known in the art. The hydrophilic fragment may contain a polyethylene glycol group, such as a methoxy-terminated polyethylene glycol group, or further connect other hydrophilic fragments via a polyethylene glycol group. The hydrophilic fragment may also contain a polyamino acid fragment, such as polyglycine or polysarcosine, which may be combined with a polyethylene glycol group. The hydrophilic fragment may also be a monosaccharide, disaccharide, or oligosaccharide, which can be a chain-like or cyclic sugar and may contain glycosamines, sugar acids, or phosphorylated sugars. Preferably, the sugar group is combined with a polyethylene glycol fragment or a polyamino acid fragment; more preferably, at least two sugar groups are introduced through a multivalent linker, such as a linker based on aspartic acid, glutamic acid, or lysine. It may also contain polycarboxylic acid groups, polysulfonic acid groups, or chelating groups, etc.

[0167] The chelating group can be, for example, a DOTA group or a NOTA group.

[0168] Unless otherwise specified, the term "heteroatom" refers to nitrogen, oxygen, sulfur, and halogen atoms.

[0169] Unless otherwise specified, the term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, a straight-chain or branched group comprising 1-20 carbon atoms, preferably comprising 1-10 carbon atoms (i.e., C10). 1-10 Alkyl groups, more preferably containing 1-8 carbon atoms (C64- ... 1-8 Alkyl groups, more preferably containing 1-6 carbon atoms (i.e., C64-C ... 1-6 Alkyl group). For example, "C 1-6 "Alkyl" refers to a group that is alkyl and has 1 to 6 carbon atoms in its carbon chain (specifically 1, 2, 3, 4, 5, or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, etc.

[0170] Unless otherwise specified, the term "alkylene" refers to a saturated straight-chain or branched divalent hydrocarbon group. For example, C1-C8 alkylene refers to a straight-chain or branched alkylene having 1-8 carbon atoms.

[0171] Unless otherwise specified, the term "heteroalkyl" means that one or more carbon atoms in an alkyl group are replaced by heteroatoms, including alkoxy groups.

[0172] Unless otherwise specified, the term "heteroalkyl" refers to a divalent "heteroalkyl" with other groups attached to both ends.

[0173] Unless otherwise specified, the term "cycloalkyl" refers to a fully saturated carbon ring that can exist as a monocyclic, bridged, or spirocyclic ring. Preferably, it contains 3-12 cyclic carbon atoms (i.e., C2C2). 3-12 cycloalkyl), more preferably containing 3-10 cyclic carbon atoms (C 3-10 Cycloalkyl groups, more preferably 3-7 cyclic carbon atoms (C 3-7 cycloalkyl groups), 4-6 cyclic carbon atoms (C 4-6 cycloalkyl groups), 5-6 cyclic carbon atoms (C 5-6 Cycloalkyl groups. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, etc.

[0174] Unless otherwise specified, the term "cycloalkylene" refers to a divalent "cycloalkyl" with other groups attached to both ends.

[0175] As used herein, the term "pharmaceutical composition" refers to a combination of at least one drug substance and optionally a pharmaceutically acceptable carrier or excipient, combined together to achieve a particular purpose. In some embodiments, the pharmaceutical composition comprises combinations that are separate in time and / or space, provided they can work together to achieve the objectives of the invention. For example, the components contained in the pharmaceutical composition (e.g., antibodies, nucleic acid molecules, combinations of nucleic acid molecules, and / or conjugates according to the invention) may be administered to an individual as a whole or separately. When the components contained in the pharmaceutical composition are administered to an individual separately, the components may be administered to the individual simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate-buffered saline), glucose, mannitol, dextran glucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or a polyalkylene glycol such as polypropylene glycol, triglycerides, etc. The type of pharmaceutically acceptable carrier used depends particularly on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The compositions according to the invention may contain wetting agents, emulsifiers, or buffering substances as additives. The pharmaceutical compositions or formulations according to the invention may be administered via any suitable route, such as oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration.

[0176] The term "therapeutic effective dose" or "effective dose" as used herein refers to a dose sufficient to demonstrate its benefit to the individual to which it is administered. The actual amount administered, as well as the rate and duration of administration, will depend on the individual's condition and severity. Prescribing treatment (e.g., determining the dosage) is ultimately the responsibility of and depends on the general practitioner and other physicians, who typically consider the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0177] EC 50 EC50 primarily refers to the concentration of a drug, antibody, or toxin that, after a specific exposure time, achieves 50% of its maximum biological effect. In pharmaceutical science, besides characterizing the activation capacity of agonists in in vitro experiments, it can also be used to indicate the blood drug concentration required to achieve half of the maximum biological effect in vivo. In some literature, EC50... 50 It is also used to characterize the potency (including agonist and antagonist) of a compound at the cellular level, and can be measured by methods such as ELISA to determine EC. 50 value.

[0178] The term "identity / homology / consistency" in relation to amino acid or nucleic acid sequences is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction, reaching the maximum percentage of identity if desired. The methods and computer programs used for alignment are well known in the art.

[0179] As used in this article, "tumor" refers to a growth or solid lesion formed by abnormal cell growth. Tumors can be benign, pre-malignant, or malignant.

[0180] The term “malignant tumor” as used in this article refers to or describes a physiological condition in mammals characterized by uncontrolled cell growth.

[0181] CrossMab technology is a bispecific antibody platform developed by Roche in Switzerland. It consists of two heterologous heavy chains and a heterologous light chain, forming a bispecific antibody structure similar to natural IgG. The Fc domain uses Knob-In-Hole technology to promote the formation of heterodimers. At the same time, the complete F(ab) or CH1-CL or VH-VL portions of one pair of light and heavy chains are interchanged, so that the light chain is precisely paired with the corresponding heavy chain.

[0182] DuetMab is a bispecific antibody platform developed by MedImmune (which has been acquired by AstraZeneca). It prevents heavy chain mismatch by using Knob-In-Hole and modifies the interchain disulfide bonds of one of the Fabs to promote the correct pairing of heavy and light chains. The specific sites are HC:F126C / C219V and LC:S121C / C214V. Attached Figure Description

[0183] Figures 1A-1C. Affinity test results of different antibodies on NCI-H1975, A431, and DIFI cells.

[0184] Figure 2. Results of endocytosis activity assay for different antibodies on NCI-H1975 cells.

[0185] Figures 3A-3B show the in vitro killing effects of ADC-1, ADC-2, and ADC-3 on DIFI and A431 cells.

[0186] Figure 4. In vitro killing effect of ADC-4, ADC-5 and ADC-6 on MKN45 cells.

[0187] Figure 5. Pharmacodynamic effect of antibody-drug conjugate on NCI-H1975 xenograft tumors in tumor-bearing mice.

[0188] Figure 6. Pharmacodynamic effect of antibody-drug conjugate on MKN45 xenografts in tumor-bearing mice.

[0189] Figure 7. Pharmacodynamic effect of antibody-drug conjugate on NCI-H441 xenograft tumors in tumor-bearing mice.

[0190] Figure 8. IgG-like structure diagram.

[0191] Figure 9. Structure diagram of IgG-scFv. Detailed Implementation

[0192] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of the present invention. The preferred embodiments and materials shown herein are for illustrative purposes only.

[0193] Any non-essential improvements and adjustments made to the implementation scheme by those skilled in the art based on the above-described invention shall still fall within the scope of protection of this application.

[0194] Example 1: Obtaining EGFR-cMET bispecific antibodies

[0195] HEK293 cells were revived and cultured in a cell culture incubator under the following conditions: 120 rpm, 8% CO2, 37°C, and a passage density of 0.3 × 10⁻⁶ cells / year. 6 Cells / mL. After two passages, plasmids were transfected, at which point the cell density was approximately 2.0 × 10 × 10⁶ cells / mL. 6 Cells / mL. Genes encoding the light and heavy chains of the antibodies were constructed into separate plasmids. During transfection, the plasmids were added in equal mass ratios: monoclonal antibodies were added at a ratio of Lc:Hc = 1:1, and penicillin antibodies at a ratio of Lc1:Hc1:Lc2:Hc2 = 1:1:1:1, with a total plasmid addition of 1 mg per liter of cells. First, the transfection buffer KPM (Zhuhai Kairui Biotechnology Co., Ltd.) was mixed with the sterile plasmids. Then, KPM was mixed with TA-293 (Zhuhai Kairui Biotechnology Co., Ltd.) to prepare the transfection reagent. The transfection reagent was slowly added to the KPM containing the plasmids, and gently mixed to prepare the plasmid-vector complex. After standing for 10 min, the plasmid-vector complex was added to the cells. 24 h later, the cell protein expression enhancer KE-293 and the transient transfection nutrient additive KT-Feed (Zhuhai Kairui Biotechnology Co., Ltd.) were added. Cells were harvested on day 6 post-transfection and purified.

[0196] Antibodies were purified using an AKTA Pure150 purifier loaded with a 1 mL Mabselect SuRe pre-packed column. First, the column was equilibrated with PBS (10 mM PB, 137 mM NaCl, pH 7.4) at a flow rate of 1 mL / min for 20 mL. Then, the sample was loaded at a flow rate of 1 mL / min. Next, 15 mL of PBS was washed at a flow rate of 1 mL / min. Finally, sodium acetate buffer (20 mM acetate-sodium acetate, pH 3.4) was used for elution at a flow rate of 1 mL / min. A 1 / 10 volume of sodium citrate neutralization solution was added to the collected sample to obtain the purified antibody. Antibodies bs2321-1a, bs2321-3c, Tilatamig, Timatamig-C8, bs2321-1a-Duet, bs3232-7, bs2321-8, and Em were prepared using this method. The amino acid sequences of the antibodies are shown below.

[0197] bs2321-1a bispecific antibody sequence

[0198] bs2321-1a First Heavy Chain and Light Chain CDR Sequences:

[0199] E-HCDR1: TYGMH (SEQ ID NO.1)

[0200] E-HCDR2:VIWDDGSYKYYGDSVKG(SEQ ID NO.2)

[0201] E-HCDR3:DGITMVRGVMKDYFDY(SEQ ID NO.3)

[0202] E-LCDR1:RASQDISSALV(SEQ ID NO.4)

[0203] E-LCDR2:DASSLES(SEQ ID NO.5)

[0204] E-LCDR3: QQFNSYPLT(SEQ ID NO.6)

[0205] BS2321-1A First Heavy Chain and Light Chain Variable Region Sequence:

[0206] E-VH:

[0207] E-VL:

[0208] >bs2321-1a First heavy chain (anti-EGFR-VH-CL-Fc)

[0209] >bs2321-1a First light chain (anti-EGFR VL-CH1)

[0210] bs2321-1a second heavy chain and light chain CDR sequences:

[0211] M-HCDR1: SYGIS (SEQ ID NO.11)

[0212] M-HCDR2: WISAYNGYTNYAQKLQG(SEQ ID NO.12)

[0213] M-HCDR3: DLRGTNYFDY(SEQ ID NO.13)

[0214] M-LCDR1:RASQGISNWLA(SEQ ID NO.14)

[0215] M-LCDR2:AASSLLS(SEQ ID NO.15)

[0216] M-LCDR3:QQANSFPIT(SEQ ID NO.16)

[0217] bs2321-1a second heavy chain and light chain variable region sequence:

[0218] M-VH:

[0219] M-VL:

[0220] >bs2321-1a Second heavy chain (anti-MET-VH-CH1-Fc)

[0221] >bs2321-1a Second light chain (anti-MET-VL-CL)

[0222] bs2321-3c bispecific antibody sequence

[0223] bs2321-3c first heavy chain and light chain CDR sequences:

[0224] E-HCDR1: NYGVH (SEQ ID NO.21)

[0225] E-HCDR2:VIWSGGNTDYNTPFTS(SEQ ID NO.22)

[0226] E-HCDR3:ALTYYDYEFAY(SEQ ID NO.23)

[0227] E-LCDR1:RASQSIGTNIH(SEQ ID NO.24)

[0228] E-LCDR2:YASESIS(SEQ ID NO.25)

[0229] E-LCDR3: QQNNNWPTT(SEQ ID NO.26)

[0230] bs2321-3c first heavy chain and light chain variable region sequence:

[0231] E-VH:

[0232] E-VL:

[0233] >bs2321-3c first heavy chain (anti-EGFR-VH-CL-Fc)

[0234] >bs2321-3c first light chain (anti-EGFR VL-CH1)

[0235] bs2321-3c second heavy chain and light chain CDR sequence:

[0236] M-HCDR1: DYYMH (SEQ ID NO.31)

[0237] M-HCDR2: RVNPNRRGTTYNQKFEG (SEQ ID NO.32)

[0238] M-HCDR3:ANWLDY(SEQ ID NO.33)

[0239] M-LCDR1:SVSSSVSSIYLH(SEQ ID NO.34)

[0240] M-LCDR2: STSNLAS(SEQ ID NO.35)

[0241] M-LCDR3: QVYSGYPLT(SEQ ID NO.36)

[0242] bs2321-3c second heavy and light chain variable region sequence:

[0243] M-VH:

[0244] M-VL:

[0245] >bs2321-3c second heavy chain (anti-MET-VH-CH1-Fc)

[0246] >bs2321-3c second light chain (anti-MET-VL-CL)

[0247] Note: CDR is determined according to the Kabat rules.

[0248] Tilatamig-C8 sequence (Tilatamig is changed from Duetmab to crossmab structure)

[0249] Tilatamig-C8 First Heavy Chain

[0250] Tilatamig-C8 First Light Chain

[0251] Tilatamig-C8 second heavy chain

[0252] Tilatamig-C8 Second Light Chain

[0253] Tilatamig sequence (antibody sequence of AZD9592, see patent CN 118556084 A)

[0254] Tilatamig First Chain

[0255] Tilatamig First Light Chain

[0256] Tilatamig second chain

[0257] Tilatamig Second Light Chain

[0258] The bs2321-1a-Duet sequence (bs2321-1a changed from a crossmab structure to a duetmab structure)

[0259] >bs2321-1a-Duet First Heavy Chain

[0260] >bs2321-1a-Duet First Light Chain

[0261] >bs2321-1a-Duet Second Chain

[0262] >bs2321-1a-Duet Second Light Chain

[0263] bs2321-7 sequence

[0264] >bs2321-7 First Heavy Chain

[0265] >bs2321-7 First Light Chain

[0266] >bs2321-7 Second chain

[0267] >bs2321-7 Second Light Chain

[0268] bs2321-8 sequence

[0269] >bs2321-8 heavy chain

[0270] >bs2321-8 light chain

[0271] GLSSPVTKSFNRGEC (SEQ ID NO.58)

[0272] bs2321-1a, bs2321-3c, Tilatamig-C8, Tilatamig, bs2321-1a-Duet, and bs2321-7 all have IgG-like structures, as shown in Figure 8; bs2321-8 has the structure shown in Figure 9.

[0273] Em (Emibetuzumab) antibody sequence

[0274] Em heavy chain

[0275] Em Light Chain

[0276] Example 2: Biomembrane Interference Determination of Antibody Affinity

[0277] The affinity of bs2321-1a, bs2321-3c, and Tilatamig bispecific antibodies for human EGFR or MET was determined using a ForteBio Octet red 96e (Sartorius) instrument. Different test samples at 5 μg / ml were immobilized using Protein A capture sensor. EGFR or MET antigens were serially diluted, starting at 100 nM and then 2-fold, for a total of 7 gradients. The buffer system was PBS pH 7.4 + 0.02% Tween 20. The procedure settings were: ① Baseline 60s, ② Loading 800s (antibody immobilization, threshold set to 0.50 nm), ③ Baseline 2 120s, ④ Association 120s, ⑤ Dissociation 300s, ⑥ Regeneration 300s. Blank buffer was used for blank treatment. The baseline was aligned with the y-axis and fitted using Savitzky-Golay data analysis software in Octet. The experimental results are shown in Tables 2 and 3.

[0278] Table 2. EGFR affinity determined by Fortebio

[0279] Table 3. MET affinity determined by Fortebio

[0280] bs2321-1a and bs2321-3c have better affinity for EGFR and MET than Tilatamig.

[0281] Example 3 Cell Binding Activity Assay

[0282] The affinities of bs2321-1a, bs2321-3c, and Tilatamig were detected using NCI-H1975 (human lung adenocarcinoma), A431 (human epidermal carcinoma), and DIFI (human colorectal cancer), respectively. Samples were diluted to 15000 ng / mL with PBS containing 2% BSA, and then serially diluted 11 times to achieve concentrations ranging from 0.014 to 15000 ng / mL. Cells were harvested, centrifuged at 500 × g for 5 min, washed three times with PBS containing 2% BSA, and incubated with different dilutions at 4°C for 2 h. After washing, cells were incubated with goat anti-human Alexa Fluor 488 fluorescent dye at 4°C in the dark for 1 h. After washing and resuspending, the mean fluorescence intensity (MFI) of cells was detected using an Attune NxT flow cytometer (Thermo Fisher Scientific, inc.). The experimental results are shown in Figures 1A-1C and Table 4.

[0283] Table 4. Affinity of the bispecific antibody to different cell types.

[0284] Example 4: Endocytosis Activity Experiment

[0285] The BS2321-1A, BS2321-3C, and Tilatamig antibodies were incubated with NCI-H1975 (human lung adenocarcinoma) at 4°C for 2 hours. After incubation, the cells were washed twice with PBS, culture medium was added, and then the cells were incubated at 37°C in a 5% CO2 incubator for endocytosis. Cells were harvested after the indicated incubation time. The cells were then stained with goat anti-human Alexa Fluor 488 and incubated at 4°C in the dark for 60 minutes. After incubation, the cells were washed twice with experimental buffer (2% FBS / PBS), resuspended in buffer, and then analyzed using flow cytometry. The median fluorescence signal of the corresponding wells in the experimental plate was read using a plate reader.

[0286] Internalization amount = Initial average median fluorescence value - Average median fluorescence value at different time points

[0287] Table 5. Ingestion of bispecific antibodies at different time points

[0288] The experimental results are shown in Figure 2 and Table 5. After bs2321-1a and bs2321-3c bind to cells, they can be rapidly internalized into cells, and the internalization amount after 6 hours is significantly higher than that of Tilatamig.

[0289] Example 5: Preparation of Antibody-Drug Conjugate (ADC)

[0290] Antibody reduction:

[0291] The antibody buffer prepared in Example 1 was replaced with PBS 7.0 / EDTA to prepare an antibody concentration of 10 mg / mL. This solution (1.0 mL) was placed into a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (equivalent to 12 equivalents per molecule of antibody) was added. The tube was incubated at 25 °C for 3 hours to reduce the interchain disulfide bonds of the antibody.

[0292] Antibody-drug-adaptor compound conjugation:

[0293] Add 68 μL of DMSO solution (10 mM deruxtecan (Shanghai Haoyuan Biomedical Technology Co., Ltd.), JSSW-001 (Shanghai Haoyuan Biomedical Technology Co., Ltd., see CN116135232B, Example 1) or SG3932 (Shanghai Haoyuan Biomedical Technology Co., Ltd., see CN118556084A) to the above solution at room temperature, mix well, and react at room temperature for 30 minutes to complete the coupling.

[0294] Purification of antibody-drug conjugates:

[0295] The above solution was purified using common operation A to obtain a solution containing the ADC-1 compound.

[0296] Determination of the drug-antibody conjugate ratio (DAR) of antibody-drug conjugates:

[0297] DAR measurements were performed using common operation B, and the DAR values ​​of ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, ADC-6, ADC-7, ADC-8, ADC-9, and ADC-10 were 7.43, 7.51, 7.56, 7.62, 7.80, 5.45, 5.40, 7.79, 7.64, and 7.82, respectively.

[0298] The structure of ADC-1 is shown in Equation II:

[0299] The ADC-2 structure is shown in Equation III:

[0300] The ADC-3 structure is shown in Equation IV:

[0301] The structure of the ADC-4 is shown in Equation V:

[0302] The ADC-5 structure is shown in Equation VI:

[0303] The ADC-6 structure is shown in Equation VII:

[0304] The ADC-7 structure is shown in Equation VIII:

[0305] The ADC-8 structure is shown in Equation IX.

[0306] The ADC-9 structure is shown in Equation X.

[0307] The structure of the ADC-10 is shown in Equation XI.

[0308] Common Procedure A: Purification of Antibody-Drug Conjugates

[0309] Equilibrate a 1 mL Mabselect SuRe pre-packed chromatography column on an AKTA system with PBS 7.0 / EDTA solution (10 mM PB, 137 mM NaCl, 5 mM EDTA, pH 7.0). Load the antibody-drug conjugate using a loading loop. After reequilibration with 10–20 mL of PBS 7.0 / EDTA, elute with acetate buffer at pH 3.5, and neutralize with 1 / 10 volume of sodium citrate neutralizing solution to obtain the purified antibody-drug conjugate.

[0310] Common Procedure B: Determination of the Drug-Antibody Conjugate Ratio (DAR) of Antibody-Drug Conjugates

[0311] Dithiothreitol was added to the antibody-drug conjugate to a final concentration of 20 mM, and the mixture was incubated in a water bath at 37°C for 30 minutes. The sample with the disulfide bonds between the antibody-drug conjugate chains cleaved was then subjected to RP-HPLC analysis. An Agilent Technologies 1260 Infinity HPLC system was used, with a PLRP-S column (5 μm particle size; 2.1 mm × 50 mm; Agilent Technologies), a column temperature of 80°C, and a detector wavelength of 280 nm. Mobile phase A was 0.1% trifluoroacetic acid (TFA) aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid (TFA) acetonitrile solution. By comparing the chromatograms of the naked antibody and the sample, light and heavy chains with different drug concentrations were distinguished. The chromatograms were integrated, and the DAR value was calculated based on the peak area.

[0312] Example 6: In vitro killing experiment of antibody-drug conjugates on DIFI and A431 cells

[0313] Target cells were collected and resuspended into single-cell suspensions. Cell viability and cell count were determined using trypan blue staining. The cell density was adjusted to 1 × 10⁶ cells / cells. 5 Cells / mL; 100 μL per well was added to a 96-well black flat-bottom cell culture plate; 20 μL of diluted test sample was added to each well of the pre-inoculated 96-well black flat-bottom cell culture plate; the plate was incubated in a cell culture incubator (37℃, 5% CO2) for 168±8 hours; 20 μL of resazurin sodium solution (0.03%) was added to each well; the plate was incubated at 37℃ for 3-4 hours, and the fluorescence values ​​were read at 550nm / 610nm using a microplate reader. The half-inhibitory concentration (IC50) of the test sample was plotted using Prism software to determine the IC50. 50 .

[0314] The experimental results are shown in Figures 3A and 3B. In the in vitro killing experiments of DIFI and A431, the killing activities of ADC-1 and ADC-2 were significantly better than those of ADC-3. The half-inhibition concentration data are summarized in Table 6.

[0315] Table 6. In vitro cytotoxic activity of antibody-drug conjugates

[0316] Example 7: In vitro killing experiment of antibody-drug conjugates on MKN45 cells

[0317] Target cells were collected and resuspended into single-cell suspensions. Cell viability and cell count were determined using trypan blue staining. The cell density was adjusted to 1 × 10⁶ cells / cells. 5 Cells / mL; 100 μL per well was added to a 96-well black flat-bottom cell culture plate; 20 μL of the diluted test sample was added to each well of the pre-inoculated 96-well black flat-bottom cell culture plate; the plate was incubated in a cell culture incubator (37℃, 5% CO2) for 168±8 hours; 20 μL of resazurin sodium solution (0.03%) was added to each well; the plate was incubated at 37℃ for 3-4 hours, and the fluorescence values ​​were read at 550nm / 610nm using a microplate reader. The half-inhibitory concentration (IC50) of the test sample was calculated using Prism or similar plotting software. 50 .

[0318] The experimental results are shown in Figure 4. In the in vitro killing experiment of MKN45, ADC-4, ADC-5, and the positive control ADC-6 all showed good killing activity. The half-inhibitory concentration data are summarized in Table 7.

[0319] Table 7. In vitro cytotoxic activity of antibody-drug conjugates

[0320] Example 8: Antitumor experiment of antibody-drug conjugate against human lung adenocarcinoma NCI-H1975 mouse xenografts

[0321] In this experiment, age-appropriate NU / NU mice were inoculated with human lung adenocarcinoma NCI-H1975 cells until the tumor volume reached approximately 110 mm. 3 Around 10:00 AM, 35 animals with good tumor growth were selected and divided into 7 groups according to tumor volume. The animal grouping and dosage regimen are shown in Table 8.

[0322] Table 8 Animal grouping and dosage regimen

[0323] After grouping, the mice were administered the drug and weighed. Data were recorded. The tumor diameter was measured at different time points after drug administration to dynamically observe tumor growth and calculate tumor volume using the following formula:

[0324] Tumor volume V = 1 / 2 × a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0325] The formula for calculating TGI is as follows:

[0326] Among them, V T t represents the average tumor volume of the test group on day t, V T 0 represents the average tumor volume of the test group on day 0, V C t represents the average tumor volume in the solvent group on day t, V C 0 represents the average tumor volume of the solvent group on day 0.

[0327] At the experimental endpoint, both ADC-4 and ADC-5 significantly inhibited tumor growth, exhibiting significantly better efficacy than the positive control ADC-6, and significantly better efficacy than ADC-8 (which has the same bispecific antibody structure) and ADC-9 (which has a monoclonal antibody structure). They were also superior to ADC-7, which has a similar antibody sequence to ADC-4 but a different linker drug. See Figure 5 and Table 9 for detailed information.

[0328] Table 9. Efficacy of antibody-drug conjugates against NCI-H1975 xenografts in tumor-bearing mice.

[0329] Example 9: Antitumor experiment of antibody-drug conjugate against human gastric cancer MKN45 mouse xenografts

[0330] In this experiment, age-appropriate NU / NU mice were inoculated with human gastric cancer MKN45 cells. When the tumor volume grew to about 100 mm3, 25 animals with good tumor growth were selected and divided into 5 groups according to the tumor volume. The animal grouping and dosage regimen are shown in Table 10.

[0331] Table 10 Animal grouping and dosage regimen

[0332] After grouping, the mice were administered the drug and weighed. Data were recorded. The tumor diameter was measured at different time points after drug administration to dynamically observe tumor growth and calculate tumor volume using the following formula:

[0333] Tumor volume V = 1 / 2 × a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0334] The formula for calculating TGI is as follows:

[0335] Among them, V T t represents the average tumor volume of the test group on day t, V T0 represents the average tumor volume of the test group on day 0, V C t represents the average tumor volume in the solvent group on day t, V C 0 represents the average tumor volume of the solvent group on day 0.

[0336] At the experimental endpoint, both ADC-4 and ADC-5 significantly inhibited tumor growth, with efficacy significantly superior to the positive controls ADC-6 and ADC-10. See Figure 6 and Table 11 for details.

[0337] Table 11. Efficacy of antibody-drug conjugates against MKN45 xenografts in tumor-bearing mice.

[0338] Example 10: Antitumor experiment of antibody-drug conjugate against human lung adenocarcinoma NCI-H441 mouse xenograft.

[0339] In this experiment, age-appropriate NU / NU mice were inoculated with human lung adenocarcinoma NCI-H441 cells until the tumor volume reached approximately 110 mm. 3 Around 10:00 AM, 20 animals with good tumor growth were selected and divided into 4 groups according to tumor volume. The animal grouping and dosage regimen are shown in Table 12.

[0340] Table 12 Animal grouping and dosage regimen

[0341] After grouping, the mice were administered the drug and weighed. Data were recorded. The tumor diameter was measured at different time points after drug administration to dynamically observe tumor growth and calculate tumor volume using the following formula:

[0342] Tumor volume V = 1 / 2 × a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0343] The formula for calculating TGI is as follows:

[0344] Among them, V T t represents the average tumor volume of the test group on day t, V T 0 represents the average tumor volume of the test group on day 0, V C t represents the average tumor volume in the solvent group on day t, V C 0 represents the average tumor volume of the solvent group on day 0.

[0345] At the experimental endpoint, both ADC-4 and ADC-5 significantly inhibited tumor growth, with significantly better efficacy than the positive control ADC-6. See Figure 7 and Table 13 for details.

[0346] Table 13. Efficacy of antibody-drug conjugates against NCI-H441 xenografts in tumor-bearing mice.

[0347] While the invention has been described herein with reference to the embodiments described above, it should be understood that the invention is not limited thereto. Those skilled in the art, and those who have received the teachings herein, will recognize other modifications and implementations that fall within the scope of this application.

Claims

1. A bispecific antibody-drug conjugate against EGFR-cMET or its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, having the structure shown in formula (I): A-(LD)d (I) Where A is a bispecific antibody against EGFR-cMET or its antigen-binding fragment, and L is a linker part, one end of which is linked to A and the other end of which is linked to the therapeutic agent D; d represents the molar ratio of the therapeutic agent to A, selected from integers or decimals from 1 to 12; the therapeutic agent is a DNA topoisomerase inhibitor.

2. The anti-EGFR-cMET bispecific antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein, -L- is selected from -L1-L2-L3-L4-, where L1 is a covalent linking unit that is covalently linked to A, L2 is an extension unit, L3 is selected from peptide residues consisting of 2-8 amino acids, and L4 is a bond or self-cleaving fragment; Preferably, L1is selected from Where * indicates a connection to A; and / or, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from -C 1-8 alkylene-, -C 1-8 Alkylene-C 3-8 Cycloalkylene-, -C 2-8 A straight-chain or branched heteroalkylene group with 1-50 (preferably 9-30, more preferably 9-26) carbon atoms, wherein the alkylene group, cycloalkylene group, and heteroalkylene group are each optionally independently selected from C16. 1-6 Alkyl, heteroalkyl with 1-6 carbon atoms, hydroxyl, amino, carboxyl or C 3-8 The heteroalkyl or heteroalkylene group is substituted by one or more substituents, and the heteroalkyl or heteroalkylene group contains 1 to 12 heteroatoms, each independently selected from N, O, or S (preferably O); more preferably, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from -C 1-6 alkylene-, -C 1-3 Alkylene-C 3-6 Cycloalkyl-,-ethynyl-C 1-6 Alkylene -, -(CH2CH2O) n -、-(CH2CH2O) n -C 1-3 alkylene-, -C 1-3 Alkylene-(CH2CH2O) n -C 1-3 alkylene-, -C 1-3 Alkylene-OC 1-3 Alkylene-, where n is an integer selected from 1 to 12; more preferably, L2 is selected from -L 2a -、-L 2a -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each is independently selected from -(CH2)5- and -CH2-O-CH2-; most preferably, L2 is selected from: -(CH2CH2O) n CH2C(O)-, -(CH2CH2O) n CH2CH2-, The * position is connected to L1; and / or L3 is selected from peptide residues consisting of 2-8 amino acids, wherein each of the 2-8 amino acids is independently selected from phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine; preferably, L3 is selected from: The * position is connected to L2; and / or, L4is selected from: a bond, -*NH-CH2-, wherein the *position is attached to L3, wherein R1is selected from hydrogen or C 1-6 alkyl.

3. The anti-EGFR-cMET bispecific antibody-drug conjugate or its tautomer, stereoisomer, or pharmaceutically acceptable salt according to claim 1 or 2, wherein, - L- is selected from The * position is connected to A.

4. The anti-EGFR-cMET bispecific antibody-drug conjugate according to any one of claims 1-3, or its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, wherein, D is selected from: wherein X is selected from the group consisting of a bond, R3 and R4 are each independently selected from H, deuterium, and C. 1-3 Alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl), 3-6 membered cycloalkyl groups (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), or R3 and R4 together with the carbon atom they are attached to form a 3-6 membered cycloalkyl group (e.g., cyclopropane, cyclobutane, cyclopentane, cyclohexane), where n' is an integer selected from 1-6 (e.g., 1, 2, 3, 4, 5, or 6), wherein the * position is connected to L4; preferably, X is selected from bonds, 5. The anti-EGFR-cMET bispecific antibody-drug conjugate according to any one of claims 1-4, or its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, wherein, D is selected from:

6. The anti-EGFR-cMET bispecific antibody-drug conjugate according to any one of claims 1-5, or its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, wherein, Formula (I) has the structure shown below: wherein R3, R4 and d are as defined in claim 4; preferably formula (I) is selected from: More preferably, formula (I) is selected from:

7. The anti-EGFR-cMET bispecific antibody-drug conjugate or its tautomer, stereoisomer, or pharmaceutically acceptable salt according to any one of claims 1-6, comprising an EGFR-targeting moiety and a cMET-targeting moiety, wherein the EGFR-targeting moiety comprises a heavy chain variable region E-VH and a light chain variable region E-VL, wherein the E-VH comprises E-HCDR1, E-HCDR2, and E-HCDR3, and the E-VL comprises E-LCDR1, E-LCDR2, and E-LCDR3, and the cMET-targeting moiety comprises a heavy chain variable region M-VH and a light chain variable region M-VL, wherein the M-VH comprises M-HCDR1, M-HCDR2, and M-HCDR3, and the M-VL comprises M-LCDR1, M-LCDR2, and M-LCDR3, wherein... (1) The sequence of E-HCDR1 includes or is the sequence shown in SEQ ID NO.1, the sequence of E-HCDR2 includes or is the sequence shown in SEQ ID NO.2, the sequence of E-HCDR3 includes or is the sequence shown in SEQ ID NO.3, the sequence of E-LCDR1 includes or is the sequence shown in SEQ ID NO.4, the sequence of E-LCDR2 includes or is the sequence shown in SEQ ID NO.5, and the sequence of E-LCDR3 includes or is the sequence shown in SEQ ID NO.6; the sequence of M-HCDR1 includes or is the sequence shown in SEQ ID NO.11, the sequence of M-HCDR2 includes or is the sequence shown in SEQ ID NO.12, the sequence of M-HCDR3 includes or is the sequence shown in SEQ ID NO.13, the sequence of M-LCDR1 includes or is the sequence shown in SEQ ID NO.14, the sequence of M-LCDR2 includes or is the sequence shown in SEQ ID NO.15, and the sequence of M-LCDR3 includes or is the sequence shown in SEQ ID NO.16; or (2) The sequence of E-HCDR1 includes or is the sequence shown in SEQ ID NO. 21; the sequence of E-HCDR2 includes or is the sequence shown in SEQ ID NO. 22; the sequence of E-HCDR3 includes or is the sequence shown in SEQ ID NO. 23; the sequence of E-LCDR1 includes or is the sequence shown in SEQ ID NO. 24; the sequence of E-LCDR2 includes or is the sequence shown in SEQ ID NO. 25; and the sequence of E-LCDR3 includes or is the sequence shown in SEQ ID NO.

26. The sequence of M-HCDR1 includes or is the sequence shown in SEQ ID NO. 31; the sequence of M-HCDR2 includes or is the sequence shown in SEQ ID NO. 32; the sequence of M-HCDR3 includes or is the sequence shown in SEQ ID NO. 33; the sequence of M-LCDR1 includes or is the sequence shown in SEQ ID NO. 34; the sequence of M-LCDR2 includes or is the sequence shown in SEQ ID NO. 35; and the sequence of M-LCDR3 includes or is the sequence shown in SEQ ID NO.

26. The sequence shown in NO.36 is determined by CDR according to the Kabat numbering rules.

8. The anti-EGFR-cMET bispecific antibody-drug conjugate of claim 7, or its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, wherein, (1) The sequence of E-VH is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 7; the sequence of E-VL is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 8; the sequence of M-VH is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 17; the sequence of M-VL is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO. 18; or (2) The sequence of E-VH is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.27, and the sequence of E-VL is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.28; the sequence of M-VH is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.37, and the sequence of M-VL is a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.

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9. The anti-EGFR-cMET bispecific antibody-drug conjugate or its tautomer, stereoisomer, or pharmaceutically acceptable salt according to any one of claims 1-8, wherein the EGFR targeting portion comprises a first heavy chain and a first light chain, the cMET targeting portion comprises a second heavy chain and a second light chain, the first heavy chain comprises a heavy chain variable region E-VH, the first light chain comprises a light chain variable region E-VL, the E-VH comprises E-HCDR1, E-HCDR2, and E-HCDR3, the E-VL comprises E-LCDR1, E-LCDR2, and E-LCDR3, the second heavy chain comprises a heavy chain variable region M-VH, the second light chain comprises a light chain variable region M-VL, the M-VH comprises M-HCDR1, M-HCDR2, and M-HCDR3, the M-VL comprises M-LCDR1, M-LCDR2, and M-LCDR3, wherein... (1) The sequence of E-HCDR1 includes or is the sequence shown in SEQ ID NO.1, the sequence of E-HCDR2 includes or is the sequence shown in SEQ ID NO.2, the sequence of E-HCDR3 includes or is the sequence shown in SEQ ID NO.3, the sequence of E-LCDR1 includes or is the sequence shown in SEQ ID NO.4, the sequence of E-LCDR2 includes or is the sequence shown in SEQ ID NO.5, and the sequence of E-LCDR3 includes or is the sequence shown in SEQ ID NO.6; the sequence of M-HCDR1 includes or is the sequence shown in SEQ ID NO.11, the sequence of M-HCDR2 includes or is the sequence shown in SEQ ID NO.12, the sequence of M-HCDR3 includes or is the sequence shown in SEQ ID NO.13, the sequence of M-LCDR1 includes or is the sequence shown in SEQ ID NO.14, the sequence of M-LCDR2 includes or is the sequence shown in SEQ ID NO.15, and the sequence of M-LCDR3 includes or is the sequence shown in SEQ ID NO.16; or (2) The sequence of E-HCDR1 includes or is the sequence shown in SEQ ID NO. 21; the sequence of E-HCDR2 includes or is the sequence shown in SEQ ID NO. 22; the sequence of E-HCDR3 includes or is the sequence shown in SEQ ID NO. 23; the sequence of E-LCDR1 includes or is the sequence shown in SEQ ID NO. 24; the sequence of E-LCDR2 includes or is the sequence shown in SEQ ID NO. 25; and the sequence of E-LCDR3 includes or is the sequence shown in SEQ ID NO.

26. The sequence of M-HCDR1 includes or is the sequence shown in SEQ ID NO. 31; the sequence of M-HCDR2 includes or is the sequence shown in SEQ ID NO. 32; the sequence of M-HCDR3 includes or is the sequence shown in SEQ ID NO. 33; the sequence of M-LCDR1 includes or is the sequence shown in SEQ ID NO. 34; the sequence of M-LCDR2 includes or is the sequence shown in SEQ ID NO. 35; and the sequence of M-LCDR3 includes or is the sequence shown in SEQ ID NO.

26. The sequence shown in NO.36 is determined by CDR according to the Kabat numbering rules.

10. The anti-EGFR-cMET bispecific antibody-drug conjugate of claim 9, or its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, wherein, (1) The sequence of E-VH includes or is the sequence shown in SEQ ID NO.7, and the sequence of E-VL includes or is the sequence shown in SEQ ID NO.8; the sequence of M-VH includes or is the sequence shown in SEQ ID NO.17, and the sequence of M-VL includes or is the sequence shown in SEQ ID NO.18; or (2) The sequence of E-VH includes or is the sequence shown in SEQ ID NO.27, and the sequence of E-VL includes or is the sequence shown in SEQ ID NO.28; the sequence of M-VH includes or is the sequence shown in SEQ ID NO.37, and the sequence of M-VL includes or is the sequence shown in SEQ ID NO.

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11. The anti-EGFR-cMET bispecific antibody-drug conjugate or a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof of any one of claims 1-10, characterized in that, The anti-EGFR-cMET bispecific antibody or its antigen-binding fragment is a chimeric, humanized, or fully human antibody or antigen-binding fragment. Preferably, the anti-EGFR-cMET bispecific antibody or its antigen-binding fragment is a fully human antibody or antigen-binding fragment.

12. The anti-EGFR-cMET bispecific antibody-drug conjugate or a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof of any one of claims 1-11, characterized in that, The antigen-binding fragment is selected from Fv, scFv, Fab, Fab', or (Fab')2.

13. The anti-EGFR-cMET bispecific antibody-drug conjugate or a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof of any one of claims 1-12, characterized in that, The anti-EGFR-cMET bispecific antibody is a full-length antibody.

14. The anti-EGFR-cMET bispecific antibody-drug conjugate of claim 13, or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, characterized in that, The anti-EGFR-cMET bispecific antibody is an IgG-like antibody, preferably an IgG1 antibody.

15. The anti-EGFR-cMET bispecific antibody-drug conjugate of claim 14, or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, characterized in that, The Fc region of the anti-EGFR-cMET bispecific antibody-drug conjugate contains amino acid mutations that reduce ADCC, CDC and / or ADCP effects, and / or Knob-Hole amino acid mutations.

16. The anti-EGFR-cMET bispecific antibody-drug conjugate of claim 15, or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, characterized in that, The amino acid mutations that reduce ADCC, CDC, and / or ADCP effects are selected from one or more amino acid mutation combinations from the following group: L234F, L235E, P329A, P326G, and P331S; in the Knob-Hole amino acid mutations, the Knob mutation is selected from one or more amino acid mutation combinations from the following group: T366W or S354C, and the Hole mutation is selected from one or more amino acid mutation combinations from the following group: T366S, L368A, Y407V, or Y349C, and the numbers are determined using the Kabat EU index.

17. The anti-EGFR-cMET bispecific antibody-drug conjugate or a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof of any one of claims 1-16, characterized in that, The first heavy chain and the first light chain of the anti-EGFR-cMET bispecific antibody exhibit CH1-CL exchange.

18. The anti-EGFR-cMET bispecific antibody-drug conjugate or a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof of any one of claims 1-17, characterized in that, The first light chain of the anti-EGFR-cMET bispecific antibody contains a λ-type light chain constant region, and the second light chain contains a κ-type light chain constant region.

19. The anti-EGFR-cMET bispecific antibody-drug conjugate or its tautomer, stereoisomer, or pharmaceutically acceptable salt according to any one of claims 1-18, characterized in that, (1) The first heavy chain includes or has the sequence shown in SEQ ID NO. 9, the first light chain includes or has the sequence shown in SEQ ID NO. 10, the second heavy chain includes or has the sequence shown in SEQ ID NO. 19, and the second light chain includes or has the sequence shown in SEQ ID NO. 20; or (2) The first heavy chain includes or has the sequence shown in SEQ ID NO.29, the first light chain includes or has the sequence shown in SEQ ID NO.30, the second heavy chain includes or has the sequence shown in SEQ ID NO.39, and the second light chain includes or has the sequence shown in SEQ ID NO.

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20. The anti-EGFR-cMET bispecific antibody-drug conjugate or its tautomer, stereoisomer, or pharmaceutically acceptable salt according to any one of claims 1-19, characterized in that, The anti-EGFR-cMET bispecific antibody-drug conjugate is ADC-1, ADC-2, ADC-3, ADC-4, or ADC-5.

21. A nucleic acid molecule encoding a bispecific anti-EGFR-cMET antibody comprising the first heavy chain, the first light chain, the second heavy chain, and the second light chain, wherein, (1) The first heavy chain includes or has the sequence shown in SEQ ID NO. 9, the first light chain includes or has the sequence shown in SEQ ID NO. 10, the second heavy chain includes or has the sequence shown in SEQ ID NO. 19, and the second light chain includes or has the sequence shown in SEQ ID NO. 20; or (2) The first heavy chain includes or has the sequence shown in SEQ ID NO.29, the first light chain includes or has the sequence shown in SEQ ID NO.30, the second heavy chain includes or has the sequence shown in SEQ ID NO.39, and the second light chain includes or has the sequence shown in SEQ ID NO.

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22. An expression vector comprising the nucleic acid molecule of claim 21.

23. A host cell comprising the expression vector of claim 22.

24. A pharmaceutical composition comprising the anti-EGFR-cMET bispecific antibody-drug conjugate as described in any one of claims 1-20, or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof.

25. The use of the anti-EGFR-cMET bispecific antibody-drug conjugate or its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 24, in the preparation of a medicament for treating an individual tumor; preferably, the tumor is an EGFR and / or cMET positive tumor; more preferably, the tumor is an EGFR and / or cMET positive solid tumor or hematologic malignancy; even more preferably, the tumor is lung cancer, gastric cancer, colorectal cancer, or skin cancer; more preferably, the lung cancer is lung adenocarcinoma.

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